We have focused on DNA complexes of restriction endonucleases in particular. Beyond their unparalleled importance as tools for the analysis and manipulation of DNA, restriction enzymes have proven remarkable model systems for studying numerous aspects of protein-nucleic acid interactions. These proteins combine high binding strength and extraordinary sequence specificity. We take advantage of the unique experimental tools we have developed to investigate the coupling of structure, thermodynamics and function of these complexes from a fresh perspective. We are currently investigating DNA complexes of the type II restriction enzyme, EcoRV. Typically restriction endonucleases can distinguish between specific recognition and nonspecific DNA sequences quite efficiently in the absence of divalent metal co-factors that are required for cleavage. At present, however, results in literature suggest that EcoRV has unusually low sequence stringency. The majority of studies performed at pH 7.5, that is optimal for the EcoRV cleavage activity, have found less than a 10-fold difference between EcoRV binding constants to the specific and nonspecific DNA sequences in the absence of divalent ions. There is, however, one measurement in the literature that contradicts this lack of recognition stringency. Additionally, X-ray crystal structures for specific and non-cognate DNA-EcoRV complexes solved in the absence of metal co-factors are noticeably different. The interface of the specific complex is essentially anhydrous with many direct DNA-protein interactions and is very different from the non-cognate complex that has a large water filled gap at the protein-DNA interface suggesting significant differences in hydration and in binding free energies between two complexes. The biochemical techniques used to measure EcoRV-DNA binding were primarily the gel mobility shift assay and a variety of fluorescent techniques, all of which are prone to artifacts. We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA binding in solution. This technique monitors only enzymatically competent complexes of the endonuclease. It does not have the limitations of gel mobility shift assay while providing same level of sensitivity. Equilibrium measurements require knowledge of association rates, in particular. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not observed for EcoRI. A significant fraction of the total enzyme, 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The slow rate of complex formation could explain the lack of specificity reported by others. We have measured the ratio of specific and nonspecific binding constants using the self-cleavage assay, providing the long incubation times necessary to achieve equilibrium. At pH 7.6, the binding constant to a 310 bp fragment is 60-fold higher than binding constant to a 30-bp nonspecific oligonucleotide. This is about an order of magnitude larger than has been typically observed. The relative specific-nonspecific binding constant, Knsp-sp, increases strongly with decreasing pH and with increasing neutral osmolyte concentration. The osmotic pressure dependence of the relative binding constant is only weakly sensitive to pH indicating that the structures of the specific and nonspecific complexes as reflected by differences in sequestered water change minimally with pH. The large osmotic dependence observed for the Knsp-sp means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are two key parameters that strongly modulate binding specificity of the EcoRV. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. The recognition specificity or activity of DNA binding proteins can be modulated by ligands or proteins that bind to one allosteric conformation in preference to others. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association. We have also further developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. The electrophoretic mobility shift assay (EMSA) is a standard and widely used tool in molecular biology for measuring DNA-protein complex formation. Many nonspecific DNA-protein complexes, however, are weak enough that they dissociate in the gel, giving smeared bands that are difficult to quantitate precisely. In order to extend the applicability of the EMSA to these labile complexes, we have investigated the effect on adding stabilizing osmolytes (such as glycerol or triethylene glycol) to the gel itself. This project is a logical continuation of our previous work on trapping DNA-protein complexes. The dissociation of complex in a gel necessarily exposes the protein and DNA surface area that was buried in the complex. The stabilizing effect of osmolytes on protein-DNA complexes can be rationalized by the exclusion of osmolytes from exposed protein and DNA surfaces. In this work, we have focused on complexes of the restriction endonuclease EcoRI with nonspecific and noncognate star sequence oligonucleotides. Our results clearly demonstrate that including the osmolyte triethylene glycol in the gel dramatically stabilizes both the weak star and nonspecific complexes of EcoRI. Without solute, both non-cognate and nonspecific complexes simply dissociate too quickly in 10% polyacrylamide gels. We showed that 30% triethylene glycol in the gel (equivalent to 4.3 osmolal) is enough to stabilize completely complexes that have dissociation constants at regular salt and pH conditions in the micromolar range. The technique can be readily used for even weaker complexes and in principle for any RNA and DNA-protein complex that is sensitive to osmotic stress. Extension of this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis is straightforward. Results are published in the Electrophoresis Journal.